Abstract
ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10.
Highlights
ADAMTS6 and ADAMTS10 are closely-related members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin Motifs) family, with ill-defined roles
We show that glycocalyx on the surface of ARPE-19A cells was dramatically altered with the depletion of ADAMTS6 and ADAMTS10, suggesting a possible mechanism for the disruption of focal adhesions and cell-cell interactions
The second mutation was inserted into the furin cleavage site (ATS6 FM), cleavage of the pro-domain is may be needed for activation of the ADAMTS6
Summary
ADAMTS6 and ADAMTS10 are closely-related members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin Motifs) family, with ill-defined roles. Recessive mutations in ADAMTS10 cause Weill-Marchesani syndrome (WMS)[1,2] associated with short stature, thickened skin and cornea, fibrotic cardiac valves and lens defects. WMS is caused by certain dominant mutations in fibrillin-1, indicating an unexpected functional relationship between ADAMTS10 and fibrillin microfibrils. Fibrillin-1 WMS mouse showed a thickened dermis, which when examined by electron microscopy contained abundant disordered microfibrils[12]. ADAMTS10 colocalises with microfibrils in superficial dermis and fibroblast cultures, and in zonules, and can interact with fibrillin-13. Fibrillin-1 binds HS at multiple sites and HS regulates its multimerization[16,17], whilst fibrillin-1 multimers enhance HS interactions[18]. We showed that fibrillin-1 TB5 domain (site of most WMS, AD and GD mutations) binds HS and can induce focal adhesions[19], and that all tested mutations disrupted this interaction[10]. We found that ADAMTS6 disrupts the HS-rich www.nature.com/scientificreports/
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